Dual Regulation of T Cell Receptor-mediated Signaling by Oncogenic Cbl Mutant 70Z*

We previously showed that an oncogenic Cbl mutant (70Z) is constitutively active in transcriptional activation of nuclear factor at activated T cells (NFAT). However, the mechanism underlying this effect remains unclear. Here we analyzed the effects of 70Z mutations at an amino-terminal loss of function site (Gly-306) and at carboxyl-terminal potential tyrosine or serine phosphorylation sites on association with signaling proteins and on NFAT activation. Mutation at Gly-306 of 70Z disrupted its association with Zap-70 and almost completely abolished its ability to induce NFAT activation under basal and ionomycin-stimulated conditions. However, mutations at potential tyrosine or serine phosphorylation sites had little effect. In fact, expression of 70Z with Tyr-700, Tyr-731, or Tyr-774 mutated to Phe increased NFAT activity in comparison with unmutated 70Z. These findings suggest that an amino terminus-mediated interaction of 70Z with Zap-70 plays a positive role and that a carboxyl terminus-mediated, phosphotyrosine-dependent interaction with their binding proteins plays a negative role in 70Z-mediated NFAT activation. In support of this notion are the observations that 70Z reduced T cell receptor-induced NFAT activation and that wild-type Cbl further inhibited this event, suggesting that both 70Z and wild-type Cbl employ a similar mechanism by which Cbl proteins dually regulate T cell receptor-mediated signaling.

Binding of antigenic peptides presented by major histocompatibility complex molecules to the T cell receptor (TCR) 1 /CD3 complex induces a rapid increase in the activities of two families of nonreceptor PTKs, i.e. the Src (Fyn and Lck) and Syk (Syk and Zap-70) families (1,2). Activation of Lck and/or Fyn leads to tyrosine phosphorylation of immunoreceptor tyrosinebased activation motifs present in the intracellular domains of CD3 and subunits, resulting in the subsequent recruitment and activation of Zap-70 and Syk. The activated PTKs in turn propagate activation signals by phosphorylating multiple intracellular proteins, eventually leading to T cell activation, lymphokine production, and proliferation. However, many aspects of the underlying signaling cascades remain unclear.
The rapid tyrosine phosphorylation of Cbl induced by TCR engagement is complemented by the observation of the physical interaction of Cbl with upstream PTKs such as Syk/Zap-70 and Fyn. Both Src family kinases such as Fyn (6, 8, 24 -26) and Syk (25,27) or Zap-70 (24,28) have been reported to associate with Cbl. The association of Zap-70 with Cbl is activation-dependent and is mediated by a Tyr(P)-binding (PTB) domain in the amino-terminal region of Cbl (28). More recently, a N(D)XY sequence was mapped in Zap-70 (Tyr-292) (29) and Syk (Y316) (30), respectively, as the Cbl PTB-binding motif. Interestingly, the Tyr-292 of Zap-70 was reported to negatively regulate T cell activation and was postulated to be a binding site for a negative regulatory protein (31,32). Consistent with this notion is the observation that Cbl binds and inhibits the kinase activity of Syk (33).
In contrast to Cbl, a mutated form of Cbl (70Z) with a 17-amino acid deletion near the amino-terminal region of a Ring finger domain is transforming in NIH3T3 fibroblasts (34,35). Recently, it was shown that 70Z binds and enhances the kinase activity of the epidermal growth factor (EGF) receptor (36) and the platelet-derived growth factor (PDGF) receptor (37). We presented data showing that 70Z is constitutively active in the transactivation of NFAT promoter (38), a critical component of the interleukin-2 gene and other cytokine genes (39,40). The 70Z-induced NFAT activation synergizes with ionomycin treatment and is dependent on a functional Ras (38). However, the exact mechanism underlying the 70Z-mediated signal leading to NFAT activation remains unclear. Exploration of this mechanism will be critical in understanding the functional role of Cbl and its oncogenic mutants in T cell activation and leukemogenesis.
Our hypothesis is that 70Z-induced NFAT transactivation is mediated via complex(es) formation of 70Z with its binding partners. To test this hypothesis, we systematically constructed a series of point-mutated 70Z mutants at an aminoterminal loss of function (Gly-306) site and at carboxyl-termi-nal potential Tyr(P) or phosphoserine sites and analyzed their interactions with respective binding partners and effects on NFAT activation. We demonstrated that expression of the G306E mutant, which disrupted the interaction with Zap-70, abolished 70Z-induced NFAT activation under both basal and ionomycin-stimulated conditions. However, mutations at Tyr-700, Tyr-731, and Tyr-774, but not at other single tyrosine or serine residues, enhanced the basal and ionomycin-treated activation of NFAT. Our results suggest that the interaction of the amino-terminal PTB domain of 70Z with Zap-70 plays a positive role and that the interactions of its carboxyl-terminal Tyr(P) residues with potential binding partners such as PI3-K and Crk-L can play a negative role in this event.
Plasmids-cDNAs encoding HA-tagged wild-type Cbl, 70Z, in pEFneo (42) were reported (38). HA-tagged 70Z subcloned into pGEM11Z was used for point mutations with a site-directed mutagenesis kit (QuickChange; Stratagene) with a pair of sense and antisense primers of 20 -23 mer around the targeting site. The mutated sequences were verified by direct DNA sequencing. The following mutations were constructed ( Fig. 1): 1) 70Z point tyrosine to phenylalanine mutations including the potential Tyr(P) residues in its carboxyl terminus (Tyr-552, Tyr-674, Tyr-700, Tyr-731, Tyr-735, and Tyr-774, single site; Tyr-700/774 and Tyr-869/871, double site) or a mutant with all eight tyrosine residues mutated (Y8F); 2) point serine to alanine mutation at the Ser-619 single site and the Ser-619/639 double site, which are within the 14-3-3 binding sites (23); and 3) a loss of function glycine to glutamic acid mutation at Gly-306. cDNAs encoding mutated 70Z constructs were subcloned back into pEFneo. The NFAT-luciferase reporter construct was provided by Dr. G. Crabtree.
Cell Culture, Transfection, and Stimulation-Simian virus 40 T an-tigen (TAg)-transfected human leukemic Jurkat T cells (Jurkat-TAg) were grown in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum and antibiotics. For protein expression in Jurkat-TAg T cells, cells were transfected with the appropriate amount of plasmids (usually 5-10 g, total) by electroporation as described previously (23). Cells were resuspended (2 ϫ 10 7 cells/ml) in 0.5 ml of medium, equilibrated at 37°C for 5 min, and activated with OKT3 (4 g/ml) for 5 min. Stimulation was terminated by adding 0.5 ml of 2ϫ Nonidet P-40 lysis buffer (2% Nonidet P-40, 40 mM Tris-HCl, pH 7.5, 300 mM NaCl, 10 mM EDTA, 10 mM sodium pyrophosphate, 10 mM NaF, 4 mM Na 3 VO 4 , and 20 g/ml each of aprotinin and leupeptin). Cells were lysed for 10 min at 4°C, and insoluble materials were removed by centrifugation at 15,000 ϫ g (4°C for 10 min). For luciferase assays, cells were washed, resuspended in RPMI 1640 medium containing 0.2% fetal calf serum, and incubated for 4 -6 h in 24-well plates. The cells were then left unstimulated or stimulated with either OKT3 ascites (1:500) or purified OKT3 (3 g/ml), ionomycin (100 ng/ml), or both ionomycin and phorbol myristate acetate (50 ng/ml) for another 8 -10 h. Immunoprecipitation and Immunoblotting-Lysates (1 ϫ 10 7 cells) were mixed with antibodies for 2 h, followed by the addition of 40 l of protein A/G Plus-Sepharose beads (Santa Cruz Biotechnology) for an additional hour at 4°C. Immunoprecipitates were washed four times with 1ϫ Nonidet P-40 lysis buffer and boiled in 30 l of 2ϫ Laemmli's buffer. Samples were subjected to SDS-10% polyacrylamide gel electrophoresis analysis and electrotransferred onto polyvinylidene difluoride membranes (Millipore). Membranes were immunoblotted with the indicated primary antibodies (usually 1 g/ml), followed by horseradish peroxidase-conjugated secondary antibodies. Membranes were washed and visualized with an ECL detection system (Amersham). When necessary, membranes were stripped by incubation in 62.5 mM Tris-HCl, pH 6.7, 100 mM 2-mercaptoethanol, and 2% SDS for 1 h at 70°C with constant agitation, washed, and then reprobed with other antibodies as indicated.
Luciferase Assay-The luciferase assay to determine the activation of reporter genes was described previously (38). Luciferase activity was determined in triplicate and expressed as arbitrary units (AU). The standard deviation among triplicates was Յ10%, and each experiment was repeated at least three times.

RESULTS
Expression of Y700F, Y731F, Y774F, or Y700/774F Enhanced 70Z-mediated NFAT Activation-We previously showed that 70Z induces the transactivation of NFAT in synergy with a Ca 2ϩ signal (38). However, the mechanism underlying this effect is unknown. To understand this mechanism, we first made several Tyr to Phe mutations at Tyr-700, Tyr-731, Tyr-774, or Tyr-700/774, which are shown to be the binding sites for Vav, PI3-K, and Crk-L (both Tyr-700 and Tyr-774), respectively (16,18,21,38), and reconfirmed their respective roles for proteinprotein interaction. As shown in Fig. 2A, the Y700F or Y774F single mutation had only a partial effect on Crk-L interaction. However, a Y700/774F double mutation completely abolished its interaction with Crk-L, indicating that both tyrosine residues are required for the Crk-L interaction (18). A Y731F mutation disrupted the interaction with p85, consistent with our previous observation (38). Under the same conditions, we did not detect Vav/70Z interaction as reported previously for Vav/Cbl interaction (21), suggesting that a Vav/70Z interaction is relatively weaker than that of 70Z with Crk-L or PI3-K.
Next we examined whether these mutants affect 70Z-mediated NFAT transactivation. Plasmids containing 70Z or its mutants were cotransfected into Jurkat-TAg cells with the NFAT-luc reporter gene. Cells were left unstimulated or stimulated with OKT3 or ionomycin and assayed for luciferase activity. As shown in Fig. 2B, 70Z induced NFAT activation under resting conditions, which synergized with ionomycin treatment, consistent with our previously published result (38). However, OKT3 stimulation did not enhance but rather reduced the luciferase activity in comparison with cells transfected with the empty vector under the same conditions, suggesting that OKT3-stimulated tyrosine phosphorylation of 70Z and its recruitment of other binding molecules may play a negative role in the TCR-mediated NFAT activation. This suggestion is supported by the observation from the point-mutated 70Z proteins. Increases in the NFAT-driven luciferase activity, albeit to slightly different degrees, were observed in cells transfected with Y700F, Y731F, Y774F, or Y700/774F under unstimulated conditions or OKT3-or ionomycin-stimulated conditions as compared with unmutated 70Z. The increases in NFAT activation were more obvious in ionomycin-treated samples. These results suggest that these tyrosine residues are not responsible for the observed ability of 70Z in NFAT transactivation. Rather, interactions of 70Z with its binding partners such as PI3-K or Crk-L via these residues may play a negative role in this event.
Although the tyrosine residues at 700, 731, and 774 are known to be responsible for the protein-protein interactions, as also shown above, there are other potential tyrosine residues whose nature of tyrosine phosphorylation or potential role in protein-protein interactions are unclear. Therefore, we made additional point mutations at Tyr-552, Tyr-674, and Tyr-735, a double mutant at Tyr-869 and Tyr-871, or a mutant with all eight tyrosine residues mutated to phenylalanine (Y8F). These mutants were expressed in Jurkat-TAg cells and analyzed for their interactions with PI3-K and Crk-L. As shown in Fig. 2C, Y552F, Y735F, or Y869/871F did not have any effect on their interactions with PI3-K and Crk-L. However, Y8F completely disrupted the interactions with PI3-K and Crk-L. A single Y674F mutant did not express well and was not included in those experiments. These mutants were then analyzed for their ability to induce NFAT transactivation. As shown in Fig. 2D, Y552F, Y735F, and Y869/831F showed only slightly enhancing effects on NFAT transactivation as compared with 70Z under unstimulated conditions or OKT3-or ionomycin-stimulated conditions. OKT3. However, Y8F, a mutant with all eight carboxyl-terminal tyrosine residues mutated, markedly enhanced its ability to activate NFAT under unstimulated or stimulated conditions. This result suggests that tyrosine residues at 552, 735, 869, and 871 are not critical for 70Z-mediated NFAT transactivation. The observed effect of Y8F may represent a synergy of the Y700F, Y731F, and Y774F mutations.
Effects of 70Z Mutants Deficient in 14-3-3 Binding on NFAT Activation-We previously demonstrated that Cbl interacts with 14-3-3 in an activation-dependent manner via two phosphoserine-containing motifs in Cbl (22,23). Of these motifs, Ser-619 and Ser-639 have been predicated to be the serine phosphorylation sites responsible for the binding (23,43). We then constructed a S619A single mutant and a S619/639A double mutant in 70Z and analyzed their interaction with 14-3-3. As shown in Fig. 3A, a mutation at Ser-619 reduced both basal and stimulation-induced interaction with 14-3-3; mutation at both the Ser-619 and Ser-639 sites abolished stimulation-induced 14-3-3 binding, although a residual basal level of 70Z/14-3-3 association remained. Next, we examined the effects of these two mutants on NFAT activation. Mutations at either single site (S619A) or double sites (S619/639A) showed only a slightly higher activation of NFAT as compared with 70Z under either unstimulated conditions or OKT3-or ionomycinstimulated conditions (Fig. 3B). Analysis of cell lysates with anti-HA revealed comparable amounts of proteins among all the samples (Fig. 3C). Although 14-3-3 may still have some effects on 70Z in these mutants because of remaining basal level interaction, this result suggests that an OKT3-stimulated, enhanced 70Z/14-3-3 interaction is not critical in 70Zinduced NFAT activation.
A Loss of Function G306E Mutant Was Critical for 70Zmediated NFAT Activation-Previous studies have shown that a point mutation (G306E) in v-Cbl, which corresponds to a loss of function mutation in Sli-1, a Caenorhabditis elegans Cbl homologue, disrupts its interaction with PTKs including Zap-70 (28) and ablates v-Cbl-induced cell transformation (37,44). Subsequent studies, including our own, demonstrated that this region contains a PTB domain that interacts with a N(D)XY motif in Zap-70 (29) and Syk (30). These observations suggest that this Cbl/PTK binding can play an important role in Cbl-mediated signaling. In support of this suggestion is the observation that Cbl inhibits Syk activity by directly binding the latter (33). To understand whether 70Z-mediated NFAT activation involves a similar mechanism, we made a G306E mutation in 70Z and transfected this mutant plasmid plus NFAT-luc into Jurkat-TAg cells. G306E completely abrogated 70Z-induced NFAT activation under resting conditions (Fig. 4). OKT3 stimulation of the cells overexpressing G306E induced further inhibition of NFAT activation by G306E as compared with 70Z. More importantly, this mutant also abolished ionomycin-induced NFAT activation by 70Z. This result provides further evidence for an evolutionarily conserved mechanism by which 70Z exerts its biological function via the interaction of its amino-terminal PTB domain with upstream PTKs, most likely Syk/Zap-70. In addition, the increased inhibition of NFAT activation by G306E under OKT3 stimulation could be explained by a Tyr(P)-mediated negative signaling, in agreement with the aforementioned observations (Fig. 2).
The G306E Mutant Showed Reduced Tyrosine Phosphorylation and Disrupted the 70Z/Zap-70 Interaction-We have previously shown that the interaction of the Cbl PTB domain with the Syk Tyr-316 residue is required for maximal tyrosine phosphorylation of Cbl (30). To further explore the mechanism underlying the G306E-mediated effect on NFAT activation, we first examined whether the G306E mutation affects its tyrosine phosphorylation. Lysates prepared from cells transfected with empty vector, HA-tagged 70Z, or G306E left unstimulated or stimulated with OKT3 were immunoblotted with anti-Tyr(P). As compared with 70Z, G306E showed a markedly reduced tyrosine phosphorylation (Fig. 5A, top panel). Reprobing of the membrane with anti-HA indicated that both 70Z and G306E were expressed at a similar level (bottom panel).
We then examined whether the reduced tyrosine phosphorylation of G306E resulted from a disruption of its interaction with Zap-70. The same cell lysates were then immunoprecipitated with anti-HA and immunoblotted with anti-Tyr(P). As shown in Fig. 5B, top panel, a ϳ70-kDa Tyr(P)-containing protein was coimmunoprecipitated with anti-HA from cells overexpressing HA-tagged 70Z under OKT3-stimulated conditions, which comigrated with Zap-70. Reprobing the same membrane with anti-Zap-70 failed to reveal Zap-70, suggesting that the nature of the 70Z/Zap-70 interaction is relatively weak. This weak interaction was consistent with the observations reported by other groups (24,29). Importantly, the same Tyr(P)-containing protein, most likely Zap-70, was not detected or was only weakly detected in anti-HA immunoprecipitates from cells overexpressing HA-tagged G306E under the same conditions. This result suggests that an evolutionarily conserved loss of function mutation in 70Z disrupts its interaction with Zap-70, which is required for the maximal tyrosine phosphorylation of 70Z.
Next we examined whether the G306E mutation has any effect on tyrosine phosphorylation and its interaction with Zap-70 under ionomycin-stimulated conditions. To this end, we coexpressed Zap-70 with 70Z or G306E in Jurkat-TAg cells. As shown in Fig. 5C, coexpression of Zap-70 with 70Z induced the tyrosine phosphorylation of 70Z under resting or ionomycinstimulated conditions. Ionomycin stimulation caused only a slight increase in the tyrosine phosphorylation of 70Z. However, the tyrosine phosphorylation of G306E was not detectable under the same conditions. We then analyzed the interaction of 70Z or G306E with Zap-70 under unstimulated or ionomycinstimulated conditions. A ϳ70 kDa Tyr(P)-containing protein comigrating with Zap-70 was coimmunoprecipitated with anti-HA from cells overexpressing HA-tagged 70Z and Zap-70. However, the interaction of G306E with Zap-70 was markedly impaired under either resting or ionomycin-stimulated conditions.
Previous studies have shown that 70Z interacts with and enhances the tyrosine phosphorylation and the kinase activity of the EGF and PDGF receptors, which is proposed to be responsible for 70Z-induced cell transformation in fibroblasts (36,37). To examine whether 70Z-induced NFAT transactivation is mediated by a similar mechanism (namely, the activation of Zap-70), we compared the tyrosine phosphorylation of Zap-70 among cells transfected with empty vector, 70Z, or G306E by analyzing anti-Zap-70 immunoprecipitates with anti-Tyr(P). As shown in Fig. 5D, top panel, almost no difference was observed among all the samples under OKT3-stimulated conditions. This result suggests that 70Z does not significantly affect the tyrosine phosphorylation and probably the kinase activity of Zap-70. In agreement with this suggestion is the observation that overexpression of 70Z or G306E did not change the amount of the tyrosine phosphorylated TCR chain coimmunoprecipitated by anti-Zap-70 (Fig. 5D, middle panel), suggesting that 70Z did not affect the ability of Zap-70 to bind to TCR. Equivalent amounts of Zap-70 were detected in all the samples (Fig. 5D, bottom panel).
Functional Role of Wild-Type Cbl in NFAT Transactivation-Cbl has been demonstrated to be a negative regulator in several mammalian systems. Recently, it was shown that it is also a negative regulator in TCR-induced AP-1 activation (45). In principle, Cbl can also be a negative regulator in TCRmediated NFAT activation because the NFAT promoter used in our studies consists of a NFAT binding site and an AP-1 binding site and requires both a Ca 2ϩ signal and a Ras-dependent signal for its activation (39,40). We have previously shown that wild-type Cbl has no effect on basal and ionomycin-stimulated NFAT activation (38). To examine whether wild-type Cbl has any effect on TCR-mediated NFAT activation, we transfected Jurkat-TAg cells with empty vector, 70Z, or wild-type Cbl plus NFAT-luc reporter plasmid and analyzed the NFAT-driven luciferase activity. As shown in Fig. 6, wild-type Cbl inhibited TCR-induced NFAT by 90%, which was stronger than 70Zmediated inhibition under the same conditions. This inhibitory role of wild-type Cbl in TCR-induced NFAT transactivation is specific, because the overexpression of Cbl had no effect or only a subtle effect on ionomycin- (Fig. 6) or ionomycin plus phorbol myristate acetate-induced NFAT activation (data not shown). This result indicates that Cbl is a negative regulator in TCRinduced NFAT activation. DISCUSSION We previously showed that oncogenic Cbl mutant 70Z induces NFAT activation in T cells in synergy with a Ca 2ϩ signal and in a functional Ras-dependent manner (38). Dissecting 70Z-mediated signaling using our established system could shed light on the biological function of Cbl proteins in T cell activation and leukemogenesis. Here we analyzed the biological role of an amino-terminal loss of function (Gly-306) site, the carboxyl-terminal potential Tyr(P), or phosphoserine sites of 70Z in the induction of NFAT, a critical component of interleukin-2 and other cytokine genes. We demonstrate that a mutation at Gly-306 abrogated 70Z-induced NFAT transactivation under both basal and ionomycin-treated conditions. However, mutations at Tyr-700, Tyr-731, Tyr-774, and Tyr-700/774, but not other Tyr to Phe or Ser to Ala mutations, increased both basal and ionomycin-treated NFAT activation. Our results suggest that 70Z plays both a positive and a negative role via its interaction with different targeting molecules.
In the present study, we clearly demonstrate that 70Z-mediated NFAT activation is mediated by its interaction with upstream PTKs, most likely Zap-70, because a loss of function mutation at Gly-306, which disrupts the interaction with Zap-70, almost completely abolished its ability to induce NFAT activation under both basal and ionomycin-stimulated conditions. This observation is consistent with the following previous findings: a corresponding mutation in Sli-1, a Cbl homologue in C. elegans, ablates Sli-1-mediated negative signaling (46); and a G306E mutant of v-Cbl disrupts its interaction with Zap-70 (28) or with EGF and PDGF receptors and abolishes its transforming ability in NIH3T3 cells (37,44). Indeed, it was shown that by binding to EGF and PDGF receptors, 70Z enhances their tyrosine phosphorylation and kinase activity, which provides a mechanistic insight into 70Z-mediated cell transformation (36,37). The observed NFAT activation by 70Z could use a similar mechanism, i.e. the activation of Zap-70 in T cells. However, 70Z does not have a significant effect on the tyrosine phosphorylation of Zap-70. Our result is consistent with the following observation in basophilic cells: cotransfection of 70Z with Syk did not affect the tyrosine phosphorylation and kinase activity of Syk (33). Although the interaction of 70Z with upstream PTKs induces a positive signal, it is possible that it exerts a different ability by binding different PTKs (Syk/Zap-70 versus EGF/PDGF receptor kinases) and/or in a different cellular context.
Recently, a N(D)XY motif in Zap-70 (Tyr-292) (29) and Syk (Tyr-316) (30) was identified to be responsible for the interaction with Cbl. Interestingly, Zap-70 Tyr-292 is a negative regulatory site; the Y292F mutant exhibits constitutive activation toward NFAT activation (31,32). Interestingly, Y292F does not show any difference in terms of tyrosine phosphorylation and kinase activity from wild-type Zap-70 (31,32). We recently observed the same result with Syk Y316F mutant. 2 Our results suggest that by binding to the same site in Zap-70 or Syk, 70Z can induce a positive signal. It seems unlikely that the positive signal induced by 70Z/Zap-70 interaction results from a direct activation of the latter; rather, it results from an indirect mechanism, e.g. by indirectly affecting the tyrosine phosphorylation of other Zap-70 substrates such as Vav, phospholipase C␥1, or LAT, which may function as positive mediators for 70Z-mediated NFAT activation. However, the possibility cannot be excluded that a weak activation of Zap-70 by 70Z, although not detectable by current biochemical methods, is sufficient for NFAT activation. In this regard, the Zap-70 Y292F mutant is not active under resting conditions, but it induces NFAT activation (31,32).
In the present study, we also demonstrate that 70Z mutants Y700F, Y731F, and Y774F enhance both basal and ionomycintreated NFAT activation, suggesting that Tyr-700, Tyr-731, and Tyr-774 play a negative role in this event, most likely via an interaction with their binding partners such as PI3-K or Crk-L. This notion is supported by the following findings: 1) overexpression of constitutively active PI3-K inhibited TCRmediated NFAT activation (47); 2) a Crk-L-C3G-Rap-1 signaling cascade is proposed to be a negative mediator for Cbl in anergic T cells (48); and 3) as shown in the present study, TCR cross-linking of 70Z-overexpressing T cells causes tyrosine phosphorylation and reduces NFAT transactivation by 70Z. In addition, the functional consequences of these mutations are specific, because mutations at other tyrosine or serine residues do not change the ability of 70Z to induce NFAT activation. Specifically, a mutation at Tyr-735, which is only four amino acids from Tyr-731, exhibits the same effect as unmutated 70Z on NFAT activation. These results suggest that a negative regulatory function of Tyr-700, Tyr-731, or Tyr-774 results from their interactions with downstream targeting molecules, e.g. PI3-K or Crk-L, not from structural changes from the Tyr to Phe mutation. However, it cannot be excluded that these tyrosine residues may bind other SH2-containing proteins, which are mediators for the observed OKT3-induced inhibition of NFAT by 70Z. Other possibilities such as the removal of 70Z from competing intracellular interactions, which may also enhance NFAT transactivation, cannot be ruled out either. In any case, it can be concluded that these tyrosine residues (Tyr-700, Tyr-731, or Tyr-774) play a negative role in 70Z-induced NFAT activation.
Taken together, our results clearly suggest a dual regulatory mechanism by which 70Z participates in TCR-mediated signaling: one by up-regulating the upstream PTKs (Syk/Zap-70) that it binds, and another by down-regulating downstream signaling via Tyr(P)-dependent interactions with its binding partners such as PI3-K and/or Crk-L (Fig. 7). The positive signal induced by 70Z/Zap-70 interaction is critical in ionomycin-stimulated NFAT activation. Mutation of the 70Z/Zap-70 interaction such as G306E disrupts this positive signal and thus abolishes ionomycin-induced NFAT activation. In the absence of a 70Z/Zap-70-induced positive signal, a Tyr(P)-dependent negative signal becomes dominant, leading to an enhanced inhibition of OKT3mediated NFAT activation. This model may also have significant implications for the biological function of wild-type Cbl. In the case of wild-type Cbl, an amino terminus-mediated interaction of wild-type Cbl with PTKs contributes to a negative regulatory function of Cbl. This model is consistent with sev-2 Z. Zhang, C. Elly, A. Altman, and Y.-C. Liu, unpublished data. eral recent studies; for example, Cbl is a negative regulator of Syk (33) via direct physical association between the two proteins, or a Cbl-mediated Crk-L-C3G-Rap1 signaling pathway is responsible for the defect in interleukin-2 production in anergic T cells (48). However, it is not known at present how the interaction of PTKs with the amino-terminal PTB domain of 70Z induces a positive signal, whereas the interaction with that of wild-type Cbl induces a negative one. Clearly, additional studies are needed to elucidate the molecular mechanism by which Cbl family proteins regulate the upstream Syk/Zap-70 family kinases with which they associate. These studies will be critical to understand the involvement of Cbl proteins in the regulation of TCR-or other cell surface receptor-mediated signaling pathways.